Methods of inhibiting ectopic calcification

ABSTRACT

The invention provides a method of inhibiting ectopic calcification in an individual. The method consists of administering to the individual a therapeutically effective amount of osteopontin or a functional fragment thereof.

This application is a divisional of application Ser. No. 09/206,576,filed Dec. 7, 1998 now U.S. Pat. No. 6,551,990.

This invention was made with government support under grant numbersHL40079-6A2 and HL18645 awarded by the National Institutes of Health andgrant number EEC9520161 awarded by the National Science Foundation. TheUnited States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of medicine and, morespecifically, to methods of inhibiting ectopic calcification.

2. Background Information

Deposition of calcium crystals in tissues other than teeth or bone,referred to as ectopic calcification, commonly occurs in associationwith renal failure, cardiovascular disease, diabetes and the agingprocess. A frequent finding in patients with renal failure, particularlythose undergoing long-term hemodialysis and unable to appropriatelyregulate serum mineral balance, is calcification of internal organs,including the lung, heart, stomach and kidneys. Less commonly,hemodialysis patients develop painful calcified skin lesions thatprogress to non-healing ulcers or gangrene and may require amputation ofthe affected limb.

Ectopic calcification is also a common complication of the implantationof bioprosthetic heart valves and is the leading cause of replacementvalve failure. Ectopic calcification also occurs in native heart valvesand blood vessels in association with atherosclerosis, diabetes andcardiovascular disease. The deposition of minerals in the vasculaturenarrows the orifices and hardens the walls of the affected valves andblood vessels, resulting in reduced blood flow to the heart andperipheral organs. Therefore, ectopic calcification increases the riskof valve failure, stroke, ischemia and myocardial infarction.

One protein that is abundant at the sites of ectopic calcification, suchas in atherosclerotic plaques and in calcified aortic valves, isosteopontin. Osteopontin has several known functions, includingpromoting cell adhesion, spreading and migration. Osteopontincolocalizes with sites of early calcification in coronaryatherosclerotic plaques and its expression increases as atherosclerosisdevelops. These findings, combined with studies showing that osteopontinhas calcium-binding properties in vitro, have led to the suggestion thatosteopontin may be involved in ectopic calcification. Previous studieshave not addressed the role of osteopontin in ectopic calcification invivo.

Ectopic calcification, if left untreated, results in increased morbidityand death. Current therapies to normalize serum mineral levels or toinhibit calcification of vascular tissues or implants are of limitedefficacy and cause unacceptable side effects.

Thus, there exists a need for an effective method of inhibiting ectopiccalcification. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The invention provides a method of inhibiting ectopic calcification inan individual. The method consists of administering to the individual atherapeutically effective amount of osteopontin or a functional fragmentthereof. The method can be used to inhibit ectopic calcificationassociated with a variety of conditions such as atherosclerosis,stenosis, restenosis, prosthetic valve replacement, angioplasty, renalfailure, tissue injury, diabetes and aging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide (SEQ ID NO: 1) and amino acid sequence (SEQID NO: 2) of human osteopontin, as described by Kiefer et al., NucleicAcids Res. 17:3306 (1989).

FIG. 2 shows the effects of osteopontin (a) on calcification of BASMC ascompared to vitronectin and fibronectin (b).

FIG. 3 shows the effects of osteopontin on alkaline phosphatase activityof BASMC (a) and phosphorous concentration in the medium (b) and theeffects of levamisole and osteopontin (OPN) on alkaline phosphatase(ALP) activity (c).

FIG. 4 shows the effects of osteopontin on calcium deposition (a),medium phosphorous concentration (b) and medium calcium concentration(c) at various initial calcium concentrations.

FIG. 5 shows the effects of recombinant osteopontin and its functionalfragments on HSMC calcium deposition (a) and the extent ofphosphorylation of recombinant osteopontin fragments by casein kinase II(b).

FIG. 6 shows the effect of phosphorylation and dephosphorylation ofosteopontin on HSMC calcification.

FIG. 7 shows the effect of various concentrations of osteopontin on HSMCcalcification.

FIG. 8 shows the time course of osteopontin inhibition of HSMCcalcification.

FIG. 9 shows the effect of osteopontin gene copy number on calcificationof valves implanted subcutaneously into mice.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to an effective method for the inhibition ofectopic calcification. Ectopic calcification commonly occurs inassociation with renal failure, cardiovascular disease, diabetes and theaging process. Ectopic calcification of the vasculature increases anindividual's risk of myocardial infarction, ischemia, stroke, dissectionafter angioplasty and heart valve failure. Ectopic calcification ofprosthetic implants, such as bioprosthetic heart valves, is the leadingcause of implant failure. Therefore, the method will reduce disease anddeath associated with ectopic calcification.

The method is based on the discovery that osteopontin is able toeffectively and specifically inhibit ectopic calcification. Therefore,ectopic calcification can be prevented or treated by administering atherapeutically effective amount of osteopontin or a functional fragmentthereof to an individual, either systemically or at the predicted orknown sites of ectopic calcification. As osteopontin is normally foundin calcified tissues and at the sites of ectopic calcification, it canbe administered with minimal toxic or immunogenic side effects.

As used herein, the term “ectopic calcification” is intended to mean theabnormal deposition of calcium crystals at sites other than bones andteeth. Ectopic calcification results in the accumulation of macroscopichydroxyapatite deposits in the extracellular matrix.

Ectopic calcification can occur in a variety of tissues and organs andis associated with a number of clinical conditions. For example, ectopiccalcification can be a consequence of inflammation or damage to theaffected tissues or can result from a systemic mineral imbalance.Commonly, ectopic calcification occurs in vascular tissue, includingarteries, veins, capillaries, valves and sinuses. Inflammation or damageto the blood vessels can occur, for example, as a result ofenvironmental factors such as smoking and high-fat diet. Inflammation ordamage can also occur as a result of trauma to the vessels that resultsfrom injury, vascular surgery, heart surgery or angioplasty. Vascularcalcification is also associated with aging and with disease, includinghypertension, atherosclerosis, diabetes, renal failure and subsequentdialysis, stenosis and restenosis.

Ectopic calcification also occurs in non-vascular tissues, such astendons (Riley et al., Ann. Rheum. Dis. 55:109-115 (1996)), skin (Evanset al., Pediatric Dermatology 12:307-310 (1997)), sclera (Daicker etal., Opthalmologica 210:223-228 (1996) and myometrium (McCluggage etal., Int. J. Gynecol. Pathol. 15:82-84 (1996)), each of which isincorporated herein by reference. In diseases resulting in systemicmineral imbalance, such as renal failure and diabetes, ectopiccalcification in visceral organs, including the lung, heart, kidney andstomach, is common (Hsu, Amer. J. Kidney Disease 4:641-649 (1997),incorporated herein by reference). Furthermore, ectopic calcification isa frequent complication of the implantation of biomaterials, prosthesesand medical devices, including, for example, bioprosthetic heart valves(Vyavahare et al., Cardiovascular Pathology 6:219-229 (1997),incorporated herein by reference). The methods of the invention areapplicable to ectopic calcification that occurs in association with allof these conditions.

The term “ectopic calcification” is not intended to refer to thecalcification that normally occurs within the bone matrix during boneformation and growth. Ectopic calcification, as used herein, is alsodistinct from abnormal calcification that occurs in renal tubules andurine that results in the formation of primarily calciumoxalate-containing kidney stones.

As used herein, the term “inhibiting,” in connection with inhibitingectopic calcification, is intended to mean preventing, retarding, orreversing formation, growth or deposition of extracellular matrixhydroxyapatite crystal deposits.

As used herein, the term “osteopontin” is intended to mean a moleculethat is able to inhibit ectopic calcification and that is recognizablysimilar to one or more molecules known in the art as osteopontin.Osteopontin is characterized as a phosphorylated sialoprotein having apredicted molecular weight of about 34 kDa. Due to high negativity,post-translational modifications and alternatively spliced isoforms,osteopontin has been reported to have an apparent molecular weight ofbetween about 44 and 85 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Giachelli et al.,Trends Cardiovasc. Med 5:88-95 (1995)). All of the post-translationallymodified forms and alternatively spliced isoforms of osteopontin areincluded within the definition of osteopontin as used herein.

Osteopontin has been identified in various species, including rat(Oldbert et al., Proc. Natl., Acad. Sci. USA 83:8819-8823 (1986)); mouse(Craig et al., J. Biol. Chem. 264:9682-9689(1989)); human (Kiefer etal., Nucleic Acids Res. 17:3306 (1989) and Young et al. Genomics7:491-502 (1990)); pig (Wrana et al., Nucleic Acids Res. 17:10119(1989)); cow (Kerr et al., Gene 108:237-243 (1991)); rabbit (Tezuka etal., Biochem. Biophys. Res. Commun. 186:911-917 (1992)); and chicken(Moore et al., Biochemistry 30:2502-2508 (1991)), each of which isincorporated herein by reference. Osteopontin from these species andosteopontin homologs from other vertebrates are included within thedefinition of osteopontin as used herein.

Osteopontin can be characterized by the presence of one or more domainsthat are conserved across known species. The conserved domains thatcharacterize osteopontin include, for example, an N-terminal signalsequence, casein kinase II phosphorylation sites, an alternativelyspliced domain, an Arg-Gly-Asp (RGD)-containing integrin-binding celladhesion domain, an Asp-rich calcium binding domain, a calcium bindinghomology domain and two heparin binding homology domains (Giachelli etal., supra (1995)). Therefore, newly identified molecules that possessone or more of these characteristic features of osteopontin are alsoincluded within the definition of osteopontin.

Osteopontin is also known in the art as bone sialoprotein I, uropontin,secreted phosphoprotein I, 2ar, 2B7 and Eta 1 (Giachelli et al., supra(1995)). The molecules encompassed by all of these terms used in the artare included within the definition of osteopontin as used herein.

The nucleotide and deduced amino acid sequence for human osteopontinhave been described by Kiefer et al., supra (1989), and are set forthherein as FIG. 1 (SEQ ID NOS: 1 and 2). The term osteopontin is intendedto include, for example, polypeptides having substantially the sameamino acid sequence as shown as SEQ ID NO: 2 and encoded bysubstantially the same nucleotide sequence as shown as SEQ ID NO: 1.

Modifications of osteopontin and its functional fragments that eitherenhance or do not greatly affect the ability to inhibit ectopiccalcification are also included within the term “osteopontin.” Suchmodifications include, for example, additions, deletions or replacementsof one or more amino acids from the native amino acid sequence ofosteopontin with a structurally or chemically similar amino acid oramino acid analog. For example, the substitution of one or morephosphorylated amino acids, such as serine or threonine residues, bynegatively charged amino acids, such as glutamic acid or aspartic acid,is contemplated. The substitution or addition of residues, such askinase phosphorylation consensus sequences, that can be phosphorylatedeither in vivo or in vitro is also contemplated. Modifications ofresidues between the native sites of phosphorylation, such as tobeneficially orient the phosphorylated residues to interact withhydroxyapatite or to reduce the distance between phosphorylation sites,is also contemplated. These modifications will either enhance or notsignificantly alter the structure, conformation or functional activityof the osteopontin or a functional fragment thereof.

Modifications that do not greatly affect the activity of osteopontin orits functional fragments can also include the addition or removal ofsugar, phosphate or lipid groups as well as other chemical derivationsknown in the art. Additionally, osteopontin or its functional fragmentscan be modified by the addition of epitope tags or other sequences thataid in its purification and which do not greatly affect its activity.

As used herein, the term “functional fragment,” in connection withosteopontin, is intended to mean a portion of osteopontin that maintainsthe ability of osteopontin to inhibit ectopic calcification. Afunctional fragment can be, for example, from about 6 to about 300 aminoacids in length, for example, from about 7 to about 150 amino acids inlength, more preferably from about 8 to about 50 amino acids in length.If desired, a functional fragment can include regions of osteopontinwith activities that beneficially cooperate with the ability to inhibitectopic calcification. For example, a functional fragment of osteopontincan include sequences that promote the ingrowth of cells, such asendothelial cells and macrophages, at the site of ectopic calcification.Similarly, a functional fragment of osteopontin can include sequences,such as the RGD-containing domain, that beneficially promote celladhesion and survival at the site of ectopic calcification.

As used herein, the term “individual” is intended to mean a human orother mammal, exhibiting, or at risk of developing, ectopiccalcification. Such an individual can have, or be at risk of developing,for example, ectopic calcification associated with conditions such asatherosclerosis, stenosis, restenosis, renal failure, diabetes,prosthesis implantation, tissue injury or age-related vascular disease.The prognostic and clinical indications of these conditions are known inthe art. An individual treated by a method of the invention can also bea candidate for, or have undergone, vascular surgery, includingprosthetic valve replacement or angioplasty. An individual treated by amethod of the invention can have a systemic mineral imbalance associatedwith, for example, diabetes, renal failure or kidney dialysis.

As used herein, the term “substantially the amino acid sequence,” inreference to an osteopontin amino acid sequence or functional fragmentthereof is intended to mean a sequence that is recognizably homologousto an osteopontin amino acid sequence and that inhibits ectopiccalcification. For example, a sequence that is substantially the same asan osteopontin sequence can have greater than about 70% homology with anosteopontin sequence, preferably greater than about 80% homology, morepreferably greater than about 90% homology.

As used herein, the term “prosthetic device” refers to a synthetic orbiologically derived substitute for a diseased, defective or missingpart of the body. As used herein, the term “bioprosthetic device” refersto a partially or completely biologically derived prosthetic device.Prosthetic devices are susceptible to ectopic calcification leading topremature failure, which can be inhibited by the methods of theinvention. A prosthetic device can be implanted or attached at varioussites of the body including, for example, the ear, eye, maxillofacialregion, cranium, limbs and heart.

The methods of the invention can advantageously be used to preventectopic calcification of prosthetic heart valves, such as an aortic oratrioventricular valve, with or without a stent. Replacement heartvalves can be made of a variety of materials, including metals, polymersand biological tissues, or any combination of these materials.Bioprosthetic valves include xenografted replacement valves frommammals, such as sheep, bovine and porcine, as well as human valves.Bioprosthetic heart valves are commonly subjected to tissue fixation andcan additionally be devitalized prior to implantation.

The invention provides a method of inhibiting ectopic calcification inan individual. The method consists of administering to the individual atherapeutically effective amount of osteopontin or a functional fragmentthereof. The method is advantageous as it employs a molecule thatnormally occurs at the site of ectopic calcification as a therapeuticagent. Therefore, the method will result in minimal toxicity,immunogenicity and side effects.

Osteopontin can be prepared or obtained by methods known in the artincluding, for example, purification from an appropriate biologicalsource or by chemical synthesis. An appropriate biological source ofosteopontin can be tissues, biological fluids or cultured cells thatcontain or express osteopontin. The presence and abundance ofosteopontin protein in a particular source can be determined, forexample, using ELISA analysis (Min et al., Kidney Int. 53:189-93 (1998),incorporated herein by reference) or immunocytochemistry (O'Brien etal., Arterioscler. Thromb. 14:1648-1656 (1994), incorporated herein byreference).

Osteopontin has been determined to be present in or expressed by kidneycells, hypertrophic chondrocytes, odontoblasts, bone cells, bone marrow,inner ear and brain cells. Osteopontin is also found in biologicalfluids, including milk and urine. Osteopontin is also present in tumors,particularly metastatic tumors and is a component of kidney stones(Butler et al., In: Principles of Bone Biology, Bilezikian et al., eds.,Academic Press, San Diego, pp. 167-181 (1996), incorporated herein byreference). Osteopontin is also produced by smooth muscle cells,macrophages and endothelial cells at the site of vascular lesions(O'Brien et al., Arterioscler. Thromb. 14:1648-1656 (1994), incorporatedherein by reference). Therefore, osteopontin can be purified from any ofthese sources using biochemical purification methods known in the art.

Osteopontin can also be obtained from the secreted medium of cells ofany of the above tissue lineages grown in culture. For example,osteopontin can be substantially purified from the conditioned medium ofsmooth muscle cell cultures as described by Liaw et al., Circ. Res.74:214-224 (1994), incorporated herein by reference.

The nucleotide sequences of osteopontin from a variety of species areknown, as described previously. Therefore, osteopontin or its functionalfragments can also be recombinantly expressed by appropriate host cellsincluding, for example, bacterial, yeast, amphibian, avian and mammaliancells, using methods known in the art. Methods for recombinantexpression and purification of peptides in various host organisms aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and inAnsubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989), both of which are incorporated herein byreference. Methods for the recombinant synthesis and purification ofosteopontin and exemplary functional fragments therefrom are described,for example, in Smith et al., J. Biol. Chem. 271:28485-28491 (1996),incorporated herein by reference.

Following recombinant synthesis and purification, osteopontin and itsfunctional fragments can be modified in a physiologically relevantmanner by, for example, phosphorylation, acylation or glycosylation,using enzymatic methods known in the art. A kinase that can be used tophosphorylate osteopontin or its functional fragments at biologicallyrelevant sites is casein kinase II, as described in Example IV. Otherserine-threonine kinases known in the art, such as protein kinase C canalso be used to phosphorylate osteopontin or its functional fragments.

The methods of the invention can be practiced using osteopontin or anyof its functional fragments that possess the activity of inhibitingectopic calcification. Fragments of osteopontin are selected, producedby methods known in the art and screened as described herein todetermine their ability to inhibit ectopic calcification.

Fragments of osteopontin can be produced, for example, by enzymatic orchemical cleavage of osteopontin. Methods for enzymatic and chemicalcleavage and for purification of the resultant protein fragments arewell known in the art (see, for example, Deutscher, Methods inEnzymology, Vol. 182, “Guide to Protein Purification,” San Diego:Academic Press, Inc. (1990), which is incorporated herein by reference).As an example, osteopontin contains a thrombin cleavage site betweenArg169 and Ser170. Either the N-terminal cleavage fragment or theC-terminal cleavage fragment of osteopontin can be used in the methodsof the invention.

Fragments of osteopontin can also be produced by chemical or recombinantsynthesis of peptides that have substantially the sequence ofosteopontin. For example, peptide libraries spanning overlappingsequences of osteopontin can be produced using methods known in the artand screened for their functional activity as described herein.Additionally, fragments corresponding to the N-terminal thrombincleavage fragment or the C-terminal thrombin cleavage fragment ofosteopontin can be recombinantly produced, as described by Smith et al.,supra 271:28485-28491 (1996) and used in the methods of the invention.

As disclosed herein, osteopontin can inhibit ectopic calcification bydirectly adsorbing to and inhibiting apatite crystal growth andformation. Therefore, functional fragments of osteopontin can beselected based on their predicted ability to bind to calcium or calciumdeposits. Regions that are contemplated to bind calcium include theaspartic acid rich sequence and the calcium binding homology domain.Therefore, a functional fragment of osteopontin can include, forexample, substantially the sequence of the aspartic-rich calcium bindingdomain, DDMDDEDDDD (SEQ ID NO: 3) or include substantially the sequenceof the calcium binding homology domain, DWDSRGKDSYET (SEQ ID NO: 4).

Additionally, as disclosed herein, phosphorylation can regulate theability of osteopontin to inhibit ectopic calcification. Therefore,functional fragments of osteopontin can be selected by the presence ofphosphorylation consensus sequences. A functional fragment ofosteopontin can to chosen to include, for example, substantially thesequence of the casein kinase II phosphorylation consensus region,SGSSEEK (SEQ ID NO: 5), or the C-terminal heparin binding homologydomain SKEEDKHLKFRISHELDSASSEVN (SEQ ID NO: 6), which contains threeconserved sites of serine phosphorylation. A functional fragment ofosteopontin can alternatively or additionally include the alternativelyspliced domain, NAVSSEETNDFKQE (SEQ ID NO: 7), which contains two sitesof serine phosphorylation. Additional sites of serine and threoninephosphorylation are described, for example, by Sorensen et al., Bioc.Biophys. Res. Comm. 198:200-205 (1994), incorporated herein byreference. A functional fragment of osteopontin can include one orseveral of these phosphorylated residues together with flanking aminoacids.

Fragments of osteopontin having the ability to inhibit ectopic activityinclude regions of the molecule that are highly conserved among species.Regions within human osteopontin with high sequence conservation arepresented, for example, in Giachelli et al., supra (1995). For example,a functional fragment can include the highly conserved sequenceSDESHHSDESDE (SEQ ID NO: 8). A functional fragment of osteopontin canalso include the conserved cell adhesion domain, DGRGDSVAYG (SEQ ID NO:9) or the heparin binding homology domain RKKRSKKFRR (SEQ ID NO: 10).

If desired, such as to optimize their functional activity, selectivity,stability or bioavailability, osteopontin or a functional fragmentthereof can be modified to include D-stereoisomers, non-naturallyoccurring amino acids, and amino acid analogs and mimetics. Examples ofmodified amino acids are presented in Sawyer, Peptide Based Drug Design,ACS, Washington (1995) and Gross and Meienhofer, The Peptides: Analysis,Synthesis. Biology, Academic Press, Inc., New York (1983), both of whichare incorporated herein by reference.

If desired, one or more phosphorylated serine or threonine residues canbe substituted by negatively charged amino acids, such as glutamic acidor aspartic acid. Such a modification can be advantageously made toreduce the susceptibility of osteopontin or a functional fragment toinactivation by phosphatases.

The ability of osteopontin or a fragment selected and prepared asdescribed above to inhibit ectopic calcification can be assayed by avariety of in vitro and in vivo assays known in the art or describedherein. For example, as described in Example I, cultured vascular cells,such as bovine aortic smooth muscle cells, form calcified deposits in atime-dependent manner when treated with calcification medium containingβ-glycerophosphate. Additionally, as described in Example III, humanvascular smooth muscle cells form calcified deposits in the presence ofelevated levels of inorganic phosphate. Other culture systems forassaying the efficacy of osteopontin or a functional fragment thereof ininhibiting ectopic calcification can be determined by those skilled inthe art. For example, osteopontin can be assayed using cells or tissuesderived from other sites in the body where ectopic calcification occursincluding, for example, viscera, skin, and endothelial cells.

The amount or extent of calcification prior to and followingadministering osteopontin or a functional fragment can be determinedusing such culture systems, either qualitatively by a visual orhistochemical assessment, or by more quantitative methods. For example,calcified deposits can be detected visually as opaque areas by lightmicroscopy and as black areas by von Kossa staining. The amount orextent of calcification can also be quantitatively assessed by themethod described by Jono et al., Arterioscler. Thromb. Vasc. Biol.17:1135-1142 (1997), incorporated herein by reference, or by using acommercially available calorimetric kit such as the Calcium Kitavailable from Sigma. Alternatively, the amount or extent ofcalcification can also be quantitatively assessed using known methods ofatomic absorption spectroscopy.

As described in Examples I and III, the calcified deposits observed incultured vascular smooth muscle cells, as assessed by histochemical,ultrastructural and electron diffraction analysis, can resemble theapatite deposits present at sites of ectopic calcification. Therefore,the ability of osteopontin or a functional fragment thereof to inhibitthe deposition of calcium by cultured cells, in comparison with avehicle or protein control, is an accurate indicator of its ability toinhibit ectopic calcification in an individual.

The ability of osteopontin or a functional fragment thereof to inhibitectopic calcification can also be tested in animal models known in theart to be reliable indicators of the corresponding human pathology. Forexample, ectopic calcification can be induced by the subcutaneous orcirculatory implantation of bioprosthetic valves, such as porcine, sheepor bovine valves, into animals as described, for example, in Vyavahareet al., supra (1997). A reduction in the amount or rate of valvecalcification by administration of osteopontin or a functional fragmentcan be detected, and is a measure of the functional activity of thepreparation.

Similarly, animal models that are reliable indicators of humanatherosclerosis, renal failure, hyperphosphatemia, diabetes, age-relatedvascular calcification and other conditions associated with ectopiccalcification are known in the art. For example, topical and systemiccalciphylaxis, calcinosis and calcergy, which are experimental models ofectopic calcification are described, for example, in Bargmann, J.Rheumatology 22:5-6 (1995), Lian et al., Calcified Tissue International,35:555-561 (1983) and Boivin et al., Cell and Tissue Res. 247:525-532(1987). An experimental model of calcification of the vessel wall isdescribed, for example, by Yamaguchi et al., Exp. Path. 25:185-190(1984).

A preferred animal model for examining ectopic calcification and theeffect of osteopontin preparations is an osteopontin-deficient mouse,described by Liaw et al., J. Clin. Invest. 101:1468-1478 (1998),incorporated herein by reference, in which, as described in Example V,ectopic calcification is enhanced compared to wild-type control animals.

Medical imaging techniques known in the art, such as magnetic resonanceimaging, X-ray imaging, computed tomography and ultrasonography, can beused to assess the efficacy of osteopontin or a functional fragmentthereof in inhibiting ectopic calcification in either a human or ananimal. For example, the presence and extent of calcium deposits withinvessels can be determined by the intravascular ultrasound imaging methoddescribed by Fitzgerald et al., Circulation 86:64-70 (1994),incorporated herein by reference. A decrease in the amount or extent ofectopic calcification can readily be identified and is indicative of thetherapeutic efficacy of osteopontin or a functional fragment thereof.

Osteopontin or its functional fragments, assayed for their functionalactivity as described above, are administered to an individual in atherapeutically effective amount to inhibit ectopic calcification.Appropriate formulations, dosages and routes of delivery foradministering osteopontin or a functional fragment are well known tothose skilled in the art and can be determined for human patients, forexample, from animal models as described previously. The dosage ofosteopontin or a functional fragment thereof required to betherapeutically effective can depend, for example, on such factors asthe extent of calcification, the site of calcification, the route andform of administration, the bio-active half-life of the molecule beingadministered, the weight and condition of the individual, and previousor concurrent therapies. The appropriate amount considered to be atherapeutically effective dose for a particular application of themethod can be determined by those skilled in the art, using the guidanceprovided herein. One skilled in the art will recognize that thecondition of the patient needs to be monitored throughout the course oftherapy and that the amount of the composition that is administered canbe adjusted accordingly.

For treating humans, a therapeutically effective amount of osteopontinor its functional fragments can be, for example, between about 10 μg/kgto 500 mg/kg body weight, for example, between about 0.1 mg/kg to 100mg/kg, or between about 1 mg/kg to 50 mg/kg, depending on the treatmentregimen. For example, if osteopontin or a functional fragment isadministered several times a day, or once a day, or once every severaldays, a lower dose would be needed than if osteopontin or a functionalfragment were administered only once, or once a week, or once everyseveral weeks. Similarly, formulations that allow for timed-release ofosteopontin would provide for the continuous release of a smaller amountof osteopontin than would be administered as a single bolus dose.

Osteopontin or a functional fragment can be delivered systemically, suchas intravenously or intraarterially, to inhibit ectopic calcificationthroughout the body. Osteopontin or a functional fragment can also beadministered locally at a site known to contain or predicted to developectopic calcification. Such a site can be, for example, anatherosclerotic plaque, a segment of artery undergoing angioplasty orthe site of prosthetic implantation. Appropriate sites foradministration of osteopontin and its functional fragments can bedetermined by those skilled in the art depending on the clinicalindications of the individual being treated and whether or not theindividual is concurrently undergoing invasive surgery.

Administration of osteopontin or a functional fragment can be achievedusing various formulations of osteopontin. If desired, osteopontin canbe administered as a solution or suspension together with apharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier can be, for example, water, sodium phosphate buffer, phosphatebuffered saline, normal saline or Ringer's solution or otherphysiologically buffered saline, or other solvent or vehicle such as aglycol, glycerol, an oil such as olive oil or an injectable organicester.

A pharmaceutically acceptable carrier can additionally containphysiologically acceptable compounds that act, for example, to stabilizeor increase the absorption of osteopontin or a functional fragment. Suchphysiologically acceptable compounds include, for example, carbohydratessuch as glucose, sucrose or dextrans; antioxidants such as ascorbic acidor glutathione; chelating agents such as EDTA, which disrupts microbialmembranes; divalent metal ions such as calcium or magnesium; lowmolecular weight proteins; lipids or liposomes; or other stabilizers orexcipients. Osteopontin can also be formulated with a material such as abiodegradable polymer or a micropump that provides for controlled slowrelease of the molecule. Additionally, osteopontin can be formulatedwith a molecule, such as a phosphatase inhibitor, that reduces orinhibits dephosphorylation of osteopontin.

Osteopontin or a functional fragment can also be expressed from cellsthat have been genetically modified to express the protein. Expressionof osteopontin from a genetically modified cell provides the advantagethat sustained localized or systemic expression of the protein canoccur, thus obviating the need for repeated administrations.

Methods for recombinantly expressing proteins in a variety of mammaliancells for therapeutic purposes are known in the art and are described,for example, in Lee et al., Transfusion Medicine II 9:91-113 (1995),which is incorporated herein by reference. Types of cells that areparticularly amenable to genetic manipulation include, for example,hematopoietic stem cells, hepatocytes, vascular endothelial cells,keratinocytes, myoblasts, fibroblasts and lymphocytes.

A nucleic acid encoding osteopontin or a functional fragment can beoperatively linked to a promoter sequence, which can provideconstitutive or, if desired, inducible expression of appropriate levelsof the encoded protein. Suitable promoter sequences for a particularapplication of the method can be determined by those skilled in the artand will depend, for example, on the cell type and the desiredosteopontin expression level.

The nucleic acid encoding osteopontin or a functional fragment thereofcan be inserted into a mammalian expression vector and introduced intocells by a variety of methods known in the art (see, for example,Sambrook et al., supra (1989); and Ausubel et al., supra (1994)). Suchmethods include, for example, transfection, lipofection, electroporationand infection with recombinant vectors. Infection with viral vectorssuch as retrovirus, adenovirus or adenovirus-associated vectors isparticularly useful for genetically modifying a cell. A nucleic acidmolecule also can be introduced into a cell using known methods that donot require the initial introduction of the nucleic acid sequence into avector.

In one embodiment of the invention, a prosthetic device can be contactedwith osteopontin or a functional fragment thereof. Contacting aprosthetic device with osteopontin or a functional fragment willeffectively prevent or reduce ectopic calcification of the prostheticdevice, preventing failure of the device and the need for prematurereplacement. The prosthetic device can be contacted with osteopontin ora functional fragment either prior to, during or following implantationinto an individual, as needed.

Osteopontin or a functional fragment can contact a prosthetic device byattaching the molecule either covalently or non-covalently to theprosthetic device. An appropriate attachment method for a particularapplication of the method can be determined by those skilled in the art.Those skilled in the art know that an appropriate attachment method iscompatible with implantation of the prosthetic device in humans and,accordingly, will not cause unacceptable toxicity or immunologicalrejection. Additionally, an appropriate attachment method will enhanceor not significantly reduce the ability of osteopontin or a functionalfragment thereof to inhibit ectopic calcification of the prostheticdevice and the surrounding tissue.

Methods for covalently attaching proteins to polymers, metals andtissues are known in the art. For example, osteopontin can be attachedto the prosthetic device using chemical cross-linking. Chemicalcross-linking agents include, for example, glutaraldehyde and otheraldehydes. Cross-linking agents that link osteopontin or a functionalfragment thereof to a prosthetic device through either a reactive aminoacid group, a carbohydrate moiety, or an added synthetic moiety areknown in the art. Such agents and methods are described, for example, inHermason, Bioconjugate Techniques, Academic Press, San Diego (1996),which is incorporated herein by reference. These methods can be used tocontact a prosthetic device with a therapeutically effective amount ofosteopontin or a functional fragment thereof.

Osteopontin can also be attached non-covalently to the prosthetic deviceby, for example, adsorption to the surface of the prosthetic device. Asolution or suspension containing osteopontin or a functional fragmentthereof, together with a pharmaceutically acceptable carrier, ifdesired, can be coated onto the prosthetic device in a therapeuticallyeffective amount.

To provide sustained delivery of osteopontin or a functional fragment, aprosthetic device can also be contacted with osteopontin or a functionalfragment thereof produced by cells attached to the prosthetic device.Such cells can be seeded onto the prosthetic device and expanded eitherex vivo or in vivo. Appropriate cells include cells that normallyproduce and secrete osteopontin including, for example, macrophages,smooth muscle cells or endothelial cells. Additionally, cells that havebeen genetically modified to produce osteopontin or a functionalfragment thereof including, for example, endothelial cells andfibroblasts, can be attached to the prosthetic device. The cells thatare attached to the prosthetic device are preferably either derived fromthe individual receiving the prosthetic implant, or from animmunologically matched individual to reduce the likelihood of rejectionof the implant.

The ability of osteopontin or a functional fragment that contacts aprosthetic device to inhibit ectopic calcification can be determined byvarious methods known in the art. One such method is to implant theprosthetic device into animals and measure calcium deposition, asdescribed in Example V, in response to administration of osteopontin ora functional fragment thereof. Either a decrease in the rate or theamount of calcium deposition at the site of the explant is indicative ofthe therapeutic efficacy of the composition.

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are also includedwithin the definition of the invention provided herein. Accordingly, thefollowing examples are intended to illustrate but not limit the presentinvention.

EXAMPLE I Calcification of Cultured Bovine Vascular Cells

This example demonstrates that calcium deposition by cultured bovineaortic smooth muscle cells is a credible model of ectopic calcification.Methods for inducing physiologically relevant calcification aredescribed. These methods can be used to assay preparations ofosteopontin and fragments thereof for their ability to inhibit ectopiccalcification.

Culture of Bovine Aortic Smooth Muscle Cells

BASMCs were obtained by a modification of the explant method originallydescribed by Ross et al., J. Cell Biol., 50:172-186 (1971), which isincorporated herein by reference. Briefly, medial tissue was separatedfrom segments of bovine thoracic aorta. Small pieces of tissue (1 to 2mm³) were loosened by a one-hour incubation in DMEM containing 4.5 g/Lof glucose supplemented with 165 U/ml collagenase type I, 15 U/mlelastase type III and 0.375 mg/mL soybean trypsin inhibitor at 37° C.Partially digested tissues were placed in 6-well plates and cultured forseveral weeks in DMEM containing 4.5 g/L of glucose supplemented with20% FBS at 37° C. in a humidified atmosphere containing 5% CO₂. Cellsthat had migrated from the explants were collected and maintained ingrowth medium (DMEM containing 15% FBS and 10 mM sodium pyruvatesupplemented with 100 U/ml of penicillin and 100 μg/ml of streptomycin).To confirm that the cells isolated from bovine aortic wall were vascularsmooth muscle cells, α-smooth muscle actin, vimentin, and calponinlevels were examined by immunofluorescence microscopy.

For immunofluorescence microscopy, BASMCS were cultured on 10-well heavyTeflon-coated microscope glass slides (Cel-Line Associates Inc., USA)for 24 hours, fixed with cold methanol, blocked with PBS containing 2%BSA and 10% normal rabbit serum, and treated with monoclonalanti-α-smooth muscle actin antibody (1A4, Sigma) and monoclonalanti-vimentin antibody (V9, Dako) diluted with PBS containing 2% BSA1:50 and 1:25, respectively. Monoclonal anti-calponin antibody (CALP),Frid et al., Dev. Biol., 153:185-193 (1992), was used without dilution.As a secondary antibody, FITC-conjugated rabbit anti-mouse IgG was usedafter dilution with PBS 1:30. Mouse non-immune IgG was used as a controlfor the primary antibody.

Greater than 95% of the cells obtained as described above were stainedwith α-smooth muscle actin, vimentin, and calponin antibodies in afilamentous pattern, indicating that the cultured cells were of vascularsmooth muscle origin. For all experiments, cells were used betweenpassages 2 and 5.

Calcium Deposition by Bovine Aortic Smooth Muscle Cells

In order to examine calcification by cultured BASMC smooth muscle cells,calcification was induced by the method described by Shioi et al.,Arterioscler Thromb Vasc Biol., 15:2003-2009 (1995), which isincorporated herein by reference. Briefly, BASMC were cultured in growthmedium for 4 days, and then switched to calcification medium (DMEM (highglucose, 4.5 g/L) containing 15% FBS and 10 mM sodium pyruvate in thepresence of 10 mM of β-glycerophosphate (unless otherwise stated), 10-7M insulin, and 50 μg/ml of ascorbic acid, supplemented with 100 U/ml ofpenicillin and 100 μg/ml of streptomycin) for 10 days. The medium wasreplaced with fresh medium twice a week. In the time course experiments,the beginning day of culture in calcification medium was defined as day0.

Calcification was assessed by a modification of the method described byJono et al., Arterioscler. Thromb. Vasc. Biol. 17:1135-1142 (1997) whichis incorporated herein by reference. Briefly, the cultures weredecalcified with 0.6 N HCl for 24 hours. The calcium content of the HClsupernatant was determined calorimetrically by the o-cresolphthaleincomplexone method (Calcium Kit, Sigma). After decalcification, thecultures were washed with phosphate-buffered saline (PBS) andsolubilized with 0.1 N NaOH/0.1% sodium dodecyl sulfate(SDS). Totalprotein content was measured with a Bio-Rad Protein Assay Kit (Bio-Rad).The calcium content of the cell layer was normalized to protein content.Phosphorus and calcium concentrations in the culture medium weremeasured by the phosphomolybdate complex method (Phosphorus Kit, Sigma)and the o-cresolphthalein complexone method (Calcium Kit, Sigma),respectively. Values were expressed as the mean +/− SEM, n=3.

BASMC treated with calcification medium containing β-glycerophosphateinitiated calcium-containing mineral deposition in a time-dependentmanner over the course of 14 days. In contrast, BASMC cultured in growthmedium lacking β-glycerophosphate did not calcify. Addition ofβ-glycerophosphate resulted in an increased phosphorus concentrationwhich correlated positively with calcium deposition in the cell layer.Conversely, calcium concentration decreased in the culture medium as thecell layer became calcified.

The effects of β-glycerophosphate on calcium deposition, phosphorusconcentration and calcium concentration in the medium weredose-dependent. Calcium deposition depended on the initial concentrationof β-glycerophosphate and was half-maximal at ˜4 mM β-glycerophosphate.Phosphorus concentration in the culture medium increased with increasingconcentrations of β-glycerophosphate over the range of 0 to 10 mM.Calcium deposition in the culture medium was inversely proportional tocalcium deposition in the cell layer.

The observed calcification was not due to spontaneous precipitation ofmineral from the media as supplementation of the culture media with upto 10 mM inorganic phosphate failed to form calcified deposits in theabsence of cells. Nor did addition of calcification media to endothelialcell cultures induce mineralization.

These results indicate that the calcification of BASMC under conditionswhich elevate inorganic phosphate in the media is a specific, cell- andmatrix-mediated event.

Morphology of BASMC Calcification

To determine whether the calcification process in the BASMC culturesrepresented a physiologic-type of mineralization, histochemical,ultrastructural, and electron diffraction analyses were performed.

Mineral deposition by BASMC cultures was assessed histochemically by vonKossa staining (30 minutes, 5% silver nitrate) and light microscopyusing the method described by Mallory, F. B., in PathologicalTechniques, Second Edition, Philadelphia, W B Saunders Co., p. 152(1942), which is incorporated herein by reference). The expression ofalkalinephosphatasee was visualized by incubatingcitrate-acetone-formaldehyde fixed cells at room temperature for 15minutes with Naphthol AS-BI Alkaline Solution (Sigma).

For ultrastructural examination by transmission electron microscopy(TEM), BASMC cells grown on plastic were fixed overnight in an aldehydesolution containing 1% glutaraldehyde and 1% paraformaldehyde bufferedwith 0.1 M sodium cacodylate buffer at pH 7.2. The cultures were thenwashed with 0.1 M sodium cacodylate buffer alone, dehydrated in a gradedseries of ethanol solutions, and infiltrated and embedded in either Taabepoxy resin or LR White acrylic resin (Marivac, Nova Scotia, Canada).The resins were polymerized for 2 days at 55° C. Samples destined forepoxy embedding were also post-fixed with potassium ferrocyanide-reduced4% osmium tetroxide to provide additional membrane contrast in theelectron microscope.

For mineral analyses by selected-area electron diffraction, othercultures were treated nonaqueously by fixing only with 100% ethanol,followed by direct embedding in resin without further processing. Onemicrometer-thick survey sections were prepared from various regions ofthe cultures and stained with Toluidine blue for examination by lightmicroscopy. Thin sections (80-100 nm) of selected regions were then cutusing a diamond knife on a Reichert Ultracut E microtome and placed onFormvar-coated nickel grids evaporated with carbon. Grid-mountedsections were stained briefly with ethanolic uranyl acetate and leadcitrate and examined using a JEOL JEM 1200EX transmission electronmicroscope operating at 60 kV. Anhydrously treated samples leftunstained were used for selected-area electron diffraction using a 100μM diffraction aperture and a camera length of 80 cm. Diffractionpatterns were analyzed and compared with synthetic apatite standards andpowder diffraction files as previously reported for bone mineral (Landiset al., J. Ultrastruc. Res., 63:188-223 (1978), incorporated herein byreference).

By light microscopy, BASMC cultures grown in growth medium showed areasof monolayer and multilayered growth typical for these types of cells.Following treatment with calcification medium for 10 days, the culturesshowed extensive deposition of mineral, predominantly in multilayeredareas. Von Kossa staining confirmed the presence of phosphate-containingmineral in these cultures. The calcification was most often observed inthe extracellular matrix between cells, and was typically morepronounced at the basal aspect of the culture. The BASMCs in thesecalcified cultures were also positive for alkaline phosphatase activity.

At 14 days of culture (10 days with β-glycerophosphate), BASMC weremonolayered or multilayered and at some locations formed nodules ofcells. Ultrastructurally, where multilayered or nodular in appearance,the cells were associated with abundant extracellular matrix rich incollagen fibrils. At sites of this extracellular matrix accumulation,cells exhibited well-developed organelles typically associated withprotein synthesis and secretion. A prominent cytoskeleton was evidencedby an extensive network of intracellular microfilaments, most likelycomposed of actin.

Whereas cells cultured without β-glycerophosphate showed no evidence ofextracellular matrix calcification, those cultured with the addedorganic phosphate source showed several morphologically distinct formsof calcification associated with the cell layer. These included roughlyspherical aggregates of calcified collagen fibrils, nodular depositswith increased mineral density at the periphery, and more diffusecalcification involving both the intra- and interfibrillar compartmentsof the extracellular matrix. At these latter sites, crystals havingsomewhat larger dimensions were observed to extend from one collagenfibril to another. Membrane-bounded matrix vesicles were also found inthe extracellular matrix. Selected-area electron diffraction ofanhydrously treated and unstained tissue sections of BASMC culturescontaining calcified deposits identified the mineral phase as apatite,showing prominent diffraction reflections (from lattice planes 002, 211,112, 300) whose indices were characteristic for this type of mineral.

Alkaline phosphatase is required for normal bone mineralization (Whyteet al. Endocr. Rev., 15:439-461 (1994)) and has been shown to berequired for calcification of osteoblast and cartilage cell cultures inresponse to β-glycerophosphate (Tenenbaum et al., Bone Mineral, 2:13-26(1987)). To determine whether alkaline phosphatase was required forcalcification in BASMCs under the conditions used in these studies,cultures were treated with the alkaline phosphatase inhibitorlevamisole, or with vehicle alone. Calcium deposition in BASMC cultureswas dose-dependently inhibited by levamisole. Half-maximal inhibitionwas observed at 5×10⁻⁵ M levamisole. Vehicle treatment had no effect.Levamisole treatment was associated with a decrease in phosphorusconcentration and maintenance of high calcium concentration in theculture medium.

These results indicate that calcification of the matrix deposited byBASMC cultures resembles the mineralization observed at sites of ectopiccalcification in regard to mineral type (apatite) and the ultrastructureof the calcified deposits. For example, mineralization occurredpredominantly in association with extracellular matrix collagen fibrilsand matrix vesicles. Similar vesicular structures have been reported incalcified atherosclerotic plaques in association with elevated alkalinephosphatase activity (Kim et al., Fed Proc, 35:156-162 (1976)).Additionally, the nodular calcifications present in the calcifying BASMCcultures indicate spherulitic crystal growth which is a commonobservation in calcified atherosclerotic plaques and valves (Kim et al.,Fed Proc, 35:156-162 (1976)).

Therefore, the calcifying BASMC cultures used in these studies are ableto create an extracellular milieu capable of mineralization similar tothe mineralization observed in calcified vascular tissues in vivo,supporting their use as a model of ectopic calcification.

EXAMPLE II Osteopontin Inhibits BASMC Calcification

This example demonstrates that osteopontin inhibits BASMC calcificationin vitro, which is a credible model of ectopic calcification in vivo.Therefore, osteopontin will be a therapeutically effective inhibitor ofectopic calcification.

Rat osteopontin was purified from the conditioned medium of rat neonatalsmooth muscle cell cultures as described by Liaw et al., supra74:214-224 (1994), which is incorporated herein by reference. Thispreparation was judged to be >95% pure based on Coomassie staining andN-terminal sequence analysis.

To examine the effect of osteopontin on BASMC-mediated calcification invitro, soluble osteopontin or vehicle alone (0.1 mM sodium citrate) wasadded to the calcifying BASMC cultures. As shown in FIG. 2 a,osteopontin at 0.05, 0.5 and 5 μg/ml dose-dependently inhibitedcalcification assessed at 10 days. For example, 0.5 μg/ml of osteopontininhibited calcification by approximately 90%, and 5 μg/ml osteopontinalmost completely inhibited calcification. In contrast, vehicle alonehad no effect (FIG. 2 a). Therefore, low concentrations of exogenouslyapplied osteopontin profoundly inhibits extracellular mineralization ina calcifying vascular cell culture system.

To exclude the possibility that contaminants in the osteopontinpreparation were responsible for the observed inhibitory effect,immunodepletion experiments were performed. Medium containing 10 μg/mlosteopontin was mixed with 20 mg/ml anti-osteopontin (OP-199) or normalgoat IgG, prepared by the method described by Liaw et al., supra (1994)and incubated for 1 hr at room temperature. 250 mg protein-A-sepharosewas added and incubated for 1 hr at room temperature. Theantibody-protein A sepharose complexes were removed by centrifugation,and the remaining supernatant diluted twenty-fold for use in thecalcification studies. Unpaired Student's t test was employed to comparegroups and a probability value (p) value less than 0.05 was consideredsignificant.

Medium containing 0.5 ug/ml rat osteopontin inhibited calcification ofthe cultures by 18 fold (5.05±0.25 μmole/mg for vehicle-treated versus0.33±0.06 μmole/mg for osteopontin-treated BASMC p=0.0023).Immunodepletion of the osteopontin solution with osteopontin antibodysignificantly reduced its inhibitory activity (0.33±0.06 μmole/mg fornonimmunodepleted sample versus 2.60±0.43 μmole/mg for anti-osteopontindepleted samples, p=0.0338). In contrast, immunodepletion with normalgoat IgG did not affect the inhibitory activity of the rat osteopontinsolution (0.49±0.10 μmole/mg for normal goat IgG-treated versus0.33±0.06 μmole/mg with no immunodepletion, p=0.2480).

These results confirm that the observed inhibition of BASMC-mediatedcalcification by the osteopontin preparation was specifically due toosteopontin, rather than due to a contaminant.

To determine the specificity and uniqueness of osteopontin's effects,two additional noncollagenous extracellular matrix RGD-containingmolecules with limited structural and functional homology toosteopontin, vitronectin and fibronectin, were tested for their abilityto inhibit BASMC-mediated calcification. Rat plasma vitronectin (SigmaImmunochemicals, USA) and bovine fibronectin (TELIOS PharmaceuticalInc., USA) were resuspended in PBS at a concentration of 0.5 mg/ml andstored frozen until use. As shown in FIG. 2 b, vitronectin (VN) andfibronectin (FN), at equimolar concentrations as were effective forosteopontin, were unable to inhibit calcium deposition. Therefore, theeffect of osteopontin on inhibiting vascular calcification is highlyspecific. Furthermore, these results indicate that the capacity ofosteopontin to modulate mineralization are unrelated to itsRGD-dependent cell adhesive functions.

Mechanism of Osteocontin Inhibition

The mechanism by which osteopontin inhibited calcification was tested.One possibility was that osteopontin might function in a manner similarto levamisole by affecting alkaline phosphatase activity, therebyinhibiting production of inorganic phosphate from β-glycerophosphate andpreventing calcium phosphate deposition.

For cellular alkaline phosphatase activity measurements, cells werecultured in calcification medium in the presence of variousconcentrations of osteopontin. Cells were washed three times with PBSand cellular proteins were solubilized with 1% Triton X-100 in 0.9% NaCland centrifuged. Supernatants were assayed for alkaline phosphataseactivity by the method described by Bessey et al., J. Biol. Chem.164:321-329 (1946), which is incorporated herein by reference. One unitwas defined as the activity producing 1 nmol of p-nitrophenol within 1minute. Protein concentrations were determined with a Bio-Rad proteinassay kit (Bio-Rad). The data were normalized to the protein content ofthe cell layer.

Treatment with osteopontin did not affect the alkaline phosphataseactivity of BASMC cultures, as shown in FIGS. 3 a and 3 c. The additionof osteopontin also did not reduce the phosphorus concentration of themedium. In contrast, levamisole dose-dependently inhibited BASMCalkaline phosphatase activity (FIG. 3 b) and reduced the phosphorusconcentration in the culture medium. These results demonstrate thatosteopontin does not act by inhibiting alkaline phosphatase activity.

The possibility that osteopontin chelates or sequesters calcium in theculture media to prevent mineralization was also tested. The initialcalcification medium was supplemented with increasing concentrations ofcalcium in the presence of osteopontin or vehicle alone. Cultures werethen allowed to calcify in the presence or absence of osteopontin over a10 day period. As shown in FIG. 4 a, increasing the calcium content ofthe medium was able to overcome the inhibitory effect of osteopontin oncalcium deposition, allowing more mineral to be deposited in the celllayer. Consistent with this, a decrease in the phosphorus content (from8.2 mM to 7.3 mM) of the culture media was noted (FIG. 4 b).

The calcium content of the media at the end of the 10 day period in thepresence of osteopontin was also measured. If osteopontin acted bysequestering calcium, it was expected that either a constant or anincreasing amount of calcium would be observed in the medium, reflectingretention of calcium in the medium by osteopontin binding. However, theopposite was observed. Calcium concentration in the culture medium wasdecreased at the end of the 10 day period compared to initial calciumconcentrations, and correlated inversely with calcium deposition(compare FIGS. 4 a and 4 c). Therefore, the inhibitory effect ofosteopontin on mineralization is calcium dependent (i.e. decreased byincreasing calcium concentrations), but is not attributable to chelationof the calcium available in the medium. This observation is consistentwith the known calcium binding properties of osteopontin. It has beenshown that about 50 molecules of calcium can be bound by osteopontin atphysiological calcium concentrations (Chen et al., J. Biol. Chem.267:24871-24878 (1992)). Hence it would require about 40 μM osteopontin(2.7 mg/ml) to chelate 2 mM calcium, which is more than 5000 times theamount of osteopontin (0.5 μg/ml) demonstrated to be effective ininhibiting vascular calcification in the assays described herein.

The ultrastructural localization of endogenous and exogenous osteopontinin the BASMC cultures was also determined using immunogold labeling tofurther characterize the mechanism of osteopontin inhibition of vascularcalcification. BASMC were cultured in calcification media for 7 days toallow mineralization to begin. Purified rat osteopontin (0.5 μg/ml) wasthen added until day 10. Cultures were preserved using aldehyde fixativefollowed by embedding in LR White acrylic resin for immunocytochemistry.Post-embedding immunolabeling was performed using osteopontin antibody(OP-199) and protein A-colloidal gold complex as described by McKee etal., Microscop. Res. And Tech., 33:141-164 (1996), which is incorporatedherein by reference. Briefly, thin (80 nm) sections of the cultures wereplaced on nickel grids and incubated for 5 min with 1% ovalbumin in PBS,followed by incubation with primary antibody for 1 hr, rinsing with PBS,blocking again with ovalbumin, and then exposure to protein A-goldcomplex for 30 min. After final rinsing with distilled water, grids wereair dried and conventionally stained with uranyl acetate and leadcitrate and viewed by transmission electron microscopy. The specificityof the OP-199 antibody has been shown previously by Western blotting(Liaw et al., supra, (1994)) and by incubations using pre-immune serumand protein A-gold complex alone.

For these immunogold labeling studies, osteopontin was omitted (vehiclealone) or added on day 7 following initiation of mineralization withβ-glycerophosphate. Under these conditions, exogenously appliedosteopontin (0.5 ug/ml) was still able to inhibit BASMC culturecalcification by 50% at day 10. A low level of endogenous osteopontinwas found in untreated, mineralizing cultures, typically in a diffusepattern in the mineralized areas. In contrast, in osteopontin-treatedcultures, gold particles were abundant at sites of calcification,typically accumulating at the margins of small calcified masses orassociating with individual crystal profiles. No gold particles wereobserved when pre-immune serum and protein A-gold complex alone wereused as controls, indicating that a direct interaction of osteopontinwith the growing apatite crystals is required for its inhibitoryfunction. Osteopontin was not observed to be associated withunmineralized matrix or cells.

The results described above demonstrate that osteopontin is able toinhibit physiological calcification mediated by vascular cells at lowconcentrations by direct binding of osteopontin to apatite crystalsurfaces and inhibition of crystal growth. Therefore, osteopontin willbe therapeutically useful in preventing and treating ectopiccalcification.

EXAMPLE III Calcification of Cultured Human Vascular Cells

This example shows that calcium deposition by cultured human smoothmuscle cells in the presence of elevated inorganic phosphate is acredible model of ectopic calcification. Methods for inducingphysiologically relevant calcification are described. These methods canbe used to assay preparations of osteopontin and fragments thereof fortheir ability to inhibit ectopic calcification.

The normal adult range of serum inorganic phosphate concentration isabout 1.0-1.5 mM. A high serum phosphate level, or hyperphosphatemia,occurs in association with various disease states including, forexample, chronic renal failure and subsequent kidney dialysis. In suchdisease states the serum inorganic phosphate levels can typically exceed2 mM. In order to model ectopic calcification associated withhyperphosphatemia and to determine the effect of osteopontin and itsfunctional fragments on such calcification, a relevant in vitro modelsystem for calcification was developed, as follows.

Human vascular smooth muscle cells (HSMC) were obtained by enzymaticdigestion as described by Ross, J. Cell Biol. 50:172-186 (1971) and Liawet al., J. Clin. Invest. 95:713-724 (1995), incorporated herein byreference. Briefly, medial tissues were separated from segments of humanaorta obtained at heart transplant surgery and autopsy, respectively.For plaque SMC, coronary atherectomy-derived tissues were obtained attime of surgery. Small pieces of tissue (1 to 2 mm³) were digestedovernight in DMEM supplemented with 165 U/ml collagenase type I, 15 U/mlelastase type III and 0.375 mg/ml soybean trypsin inhibitor at 37° C.Single cell suspensions were placed in 6-well plates and cultured forseveral weeks in DMEM supplemented with 20% FCS at 37° C. in ahumidified atmosphere containing 5% CO₂. Cultures that formed colonieswere collected at confluence and maintained in growth medium (DMEMcontaining 15% FBS and 1 mM sodium pyruvate supplemented with 100 U/mlof penicillin and 100 mg/ml of streptomycin; final inorganic phosphateconcentration was 1.4 mM). Purity of cultures was assessed by positiveimmunostaining for alpha-SM actin and calponin and absence of vonWillebrand factor staining as described by Ross, supra (1971) and Liawet al., supra (1995).

Primary human adult and fetal aortic medial and coronary plaque primarycells up to passage 8 were used in these experiments. A fetal and adultHSMC culture was also immortalized using HPV-E6E7 and characterized asdescribed by Perez et al., Proc. Natl. Acad. Sci. USA 89:1224-1228(1992), incorporated herein by reference.

HSMC were routinely subcultured in growth medium. At confluence, cellswere switched to calcification medium (DMEM containing 15% FBS and 1 mMsodium pyruvate in the presence of 2 mM inorganic phosphate supplementedwith 100 U/ml penicillin and 100 μg/ml of streptomycin) for up to 14days. The medium was replaced with fresh medium every 2 days. Fortime-course experiments, the first day of culture in calcificationmedium was defined as day 0. Calcium deposition was quantified andassessed histochemically and cytochemically as described above inExample I.

In medium containing normal serum phosphate levels (inorganic phosphate,P_(i), of 1.4 mM), HSMC accumulated very little calcium mineral. Incontrast, in the presence of 2 mM inorganic phosphate, calciumdeposition increased in a time-dependent manner. For example, on day 9,calcified HSMC vs. uncalcified control was 210.3+/−2.4 vs. 15.1+/−2.4(μg/mg protein), mean +/−SEM (n+3)). The effect of inorganic phosphateon calcium deposition was dose-dependent over the range of 1.4 mM to 2mM inorganic phosphate. Induction of calcification by elevated inorganicphosphate appeared to be a general feature of human cells, since primaryHSMC derived from different sources (human adult and fetal aortic andcoronary plaque) as well as immortalized derivatives of these cellsshowed similar behavior. No spontaneous deposition of calcium mineraloccurred in calcification medium or in medium supplemented with up to 10mM inorganic phosphate, indicating that cells and/or cell-derived matrixis necessary for mineralization.

To determine whether the observed calcification in the human cellculture system was physiologically relevant, morphological studies wereperformed. After culturing HSMC in calcification medium for 10 days,granular deposits developed throughout the cell culture. The depositswere identified as phosphate-containing mineral by von Kossa staining,as described in Example I. Black-stained particles were diffuselyscattered throughout the cell layer, predominantly in the extracellularregions, with greatest accumulation in multilayered foci. Electronmicroscopic analysis confirmed the presence of an apatite mineral phase,calcified collagen fibrils and matrix vesicles associated withmineralized cultures, essentially identical to the calcification ofbovine SMC cultures in the system described in Example I.

These results demonstrate that HSMC cultures are susceptible tocalcification when cultured in media containing inorganic phosphateconcentrations typically found in hyperphosphatemic individuals.Furthermore, the observed calcification in the cultured human cells issimilar to the ectopic calcification observed in calcified tissues invivo. Therefore, the HSMC calcification culture system can be used toaccurately assess the effect of regulators of ectopic calcification.

EXAMPLE IV Inhibition of Calcification of Human Vascular Cells byOsteopontin and Functional Fragments thereof

This example demonstrates that osteopontin and exemplary functionalfragments of osteopontin effectively inhibit human smooth muscle cellcalcification. Therefore, osteopontin can be used therapeutically toinhibit ectopic calcification.

Osteopontin proteins and functional fragments were assayed for theirability to inhibit ectopic calcification using the HSMC calcificationsystem described in Example III. The osteopontin proteins includefull-length human recombinant osteopontin as well as recombinantN-terminal and C-terminal human osteopontin fragments similar to thosethat would be formed following thrombin cleavage of the native protein,as described by Smith et al., supra (1996). Two N-terminal fragmentswere used, 10N and 30N, which refer to two differ splice variants ofosteopontin. The 30N splice variant contains an additional 14 aminoacids, NAVSSEETNDFKQE (SEQ ID NO: 7), which correspond to exon 5 (aminoacids 59-72). The ION fragment contains amino acids 17-58 and 73-160 ofnative osteopontin, whereas the 30N fragment contains amino acids17-169. The 10C fragment contains the C-terminal domain of osteopontin,amino acids 170-317.

The N- and C-terminal recombinant osteopontin fragments were expressedas fusion proteins with GST, purified from bacterial lystates byaffinity chromatography on glutathione beads, and cleaved with thrombin.The full-length human recombinant osteopontin was prepared as aHis-tagged protein. The size and purity of the resulting recombinantproteins was confirmed by SDS-PAGE analysis (Smith et al., supra(1996)).

Recombinant osteopontin and its functional fragments were assayed fortheir ability to inhibit ectopic calcification of human smooth musclecells (HSMC) either prior to or following phosphorylation by caseinkinase II. The amount of phosphate incorporated into osteopontin (OPN)and its fragments achieved by casein kinase II phosphorylation is shownin FIG. 5 b. As shown in FIG. 5 a, in the presence of high-phosphatecalcification medium, calcium deposition into HSMC matrix is reduced tobasal levels by the addition of phosphorylated OPN, 30N OPN, 10N OPN or10C OPN. The non-phosphorylated forms of these proteins do notsignificantly affect calcium deposition in this assay. These resultsshow that both N- and C-terminal fragments of osteopontin are functionalfragments of osteopontin, and that serine-threonine phosphorylationappears to be important for the functional activity of osteopontin andits functional fragments.

As shown in FIG. 6, recombinant osteopontin phosphorylated by caseinkinase II is able to inhibit HSMC calcification at a concentration of 15nM. Dephosphorylation with alkaline phosphatase (ALP) reverses thisinhibitory ability. These results confirm the importance ofphosphorylation for the functional activity of osteopontin and itsfunctional fragments.

The effect of human osteopontin on inhibiting ectopic calcification isdose-dependent over the range of concentrations of 0.1 μg/ml to 5.0μg/ml (FIG. 7). Furthermore, the effect of osteopontin on inhibiting andreversing ectopic calcification is rapid, with significantly reducedcalcium deposition being apparent by 60 minutes, with approximately 50%inhibition observable by 90 minutes following addition (FIG. 8).

These results indicate that osteopontin and exemplary functionalfragments thereof are able to effectively inhibit physiologicallyrelevant ectopic calcification of human cells rapidly and at lowconcentrations. Therefore, full-length osteopontin and functionalfragments thereof will be therapeutically effective in inhibitingectopic calcification in individuals exhibiting or at risk of exhibitingectopic calcification.

EXAMPLE V Osteopontin Inhibits Ectopic Calcification in vivo

This example shows that osteopontin inhibits ectopic calcification invivo.

The effect of subcutaneous implantation of porcine prosthetic valves innormal mice and mice deficient in osteopontin was tested to determinethe role of osteopontin in ectopic calcification in vivo. Mice deficientin one or both copies of the osteopontin gene are described in Liaw etal., supra (1998). A 5.0 cm³ piece of porcine glutaraldehyde-fixedaortic valve leaflet was subcutaneously implanted into 5-6 week oldfemale mice carrying either the wild type (WT), heterozygote (HTZ) ornull allele (KO) for osteopontin. After 14 days, implants were removed,freeze-dried and acid hydrolyzed. Calcium levels were assayed asdescribed in Example I and normalized to the dried weight of theexplant.

As shown in FIG. 9, implanted valves calcify to a significantly greaterextent in osteopontin null mice than in wild-type or heterozygous mice.Therefore, consistent with the observed ability of osteopontin toinhibit ectopic calcification in relevant in vitro systems, theseresults indicate that osteopontin inhibits ectopic calcification invivo.

The foreign body inflammatory response also appears to be impaired inthe osteopontin null mouse. For example, there is an apparent reductionin infiltration by macrophages at the site of valve implantation in theosteopontin null mouse compared to the wild-type or heterozygous mice.Macrophages that normally infiltrate a site of inflammation and ectopiccalcification are contemplated to promote removal of calcified depositsby phagocytosis. Therefore, it is contemplated that osteopontin bothinhibits hydroxyapatite formation and promotes phagocytotic resorptionof calcified deposits by macrophages.

Accordingly, the administration of osteopontin or its functionalfragments to an individual will be therapeutically effective ininhibiting ectopic calcification.

Throughout this application various publications have been referencedwithin parentheses. The disclosure of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention applies.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A method of inhibiting ectopic calcification in an individual,comprising administering to said individual a therapeutically effectiveamount of osteopontin or a functional fragment thereof, wherein saidosteopontin or functional fragment is produced by endothelial cells ormacrophages attached to a prosthetic device at a site of ectopiccalcification, and wherein said amount of osteopontin or functionalfragment is sufficient to inhibit ectopic calcification in saidindividual.
 2. The method of claim 1, wherein said cells recombinantlyproduce osteopontin or a functional fragment thereof.